Eur J Clin Microbiol Infect Dis (2008) 27:1125–1129 DOI 10.1007/s10096-008-0553-z
CONCISE ARTICLE
Human antimicrobial peptides’ antifungal activity against Aspergillus fumigatus A. Lupetti & J. T. van Dissel & C. P. J. M. Brouwer & P. H. Nibbering
Received: 18 January 2008 / Accepted: 12 May 2008 / Published online: 20 June 2008 # The Author(s) 2008
Abstract In light of the need for new antifungals, we compared the in vitro antifungal activity of two peptides derived from human lactoferrin (hLF), i.e., hLF(1–11) and hLF(21–31), two analogs of histatin 5, further referred to as dhvar4 and dhvar5, and two ubiquicidin (UBI)-derived peptides, i.e., UBI 18–35 and UBI 29–41, with that of amphotericin B against Aspergillus fumigatus hyphae using the MTT assay. The results revealed a dose-dependent antifungal activity for all peptides, with dhvar5 being the most potent peptide. In addition, hLF(1–11), dhvar5, and UBI 18–35 were effective against A. fumigatus conidia. Furthermore, hLF(1–11) did not lyze human erythrocytes, whereas dhvar5 (≥16 μM) and UBI 18–35 (≥20 μM) were hemolytic. Based on these in vitro results and their effectiveness against infections in mice, we concluded that hLF(1–11) and dhvar5 are promising candidates for the development of new agents against A. fumigatus infections.
A. Lupetti : J. T. van Dissel : P. H. Nibbering Department of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 Leiden, The Netherlands A. Lupetti (*) Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Sezione di Microbiologia e Virologia, Università di Pisa, Via S. Zeno 37–39, Pisa 56127, Italy e-mail:
[email protected] C. P. J. M. Brouwer AM-Pharma BV, Bunnik, The Netherlands
Introduction A large surveillance in the United States from 1980 through 1997 revealed that, of the deaths in which an infectious disease was the underlying cause, the mortality due to invasive mycoses increased from the tenth most common cause in 1980 to the seventh most common in 1997, with an impressive 3.6-fold increase in mortality associated with aspergillosis [1]. Furthermore, a systematic review of the literature revealed a case–fatality rate of 58% for patients with invasive aspergillosis and was especially high in bone marrow transplant recipients (87%) [2]. Moreover, amphotericin B deoxycholate and lipid formulations thereof failed to prevent death in one-half to two-thirds of these patients [2]. Despite the introduction of several new antifungals (newer generation azoles and echinocandins), the mortality and morbidity of aspergillosis is still high [2, 3]. The availability of effective strategies to improve the outcome for patients with opportunistic mycoses, in particular, invasive aspergillosis, constitutes an enormous medical need and antimicrobial peptides [4–6] may be promising candidates for this purpose because their mechanisms of action differ from that of current antifungals and the acquisition of resistant mutants is less likely to occur. An interesting approach is the use of synthetic peptides derived from natural antimicrobial proteins/peptides with antifungal activities. For example, human lactoferrin (hLF), which is a protein synthesized and released by activated neutrophils and mucosal epithelial cells, exerted antifungal activities. This antimicrobial protein contains two cationic domains in its N terminus. A recent study indicated that a synthetic peptide including the first cationic domain, further referred to as hLF(1–11), is more effective in killing Candida albicans than the native protein or a peptide, hLF(21–31), comprising the second cationic domain [7].
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Moreover, the hLF(1–11) peptide is effective against an invasive infection with fluconazole-resistant C. albicans in mice [6]. Histatin 5 is a histidine-rich, cationic peptide secreted by human parotid and submandibular glands [8]. From this peptide, several analogs with antifungal activities, further referred to as dhvar4 and dhvar5, have been designed [9]. Furthermore, our research has focused on ubiquicidin, a 6,648-Da linear cationic antimicrobial peptide produced by IFN-γ-activated murine macrophages and human airway epithelial cells [10]. This peptide displays antimicrobial activity against a variety of bacteria and C. albicans species. Recently, synthetic peptides derived from ubiquicidin, e.g., UBI 18–35, and UBI 29–41, have been selected on the basis of their antimicrobial activities [11]. In a first attempt to select the most promising candidates, we compared the in vitro antifungal activities of these six synthetic antimicrobial peptides against Aspergillus fumigatus and their hemolytic effects.
Materials and methods Fungal strain and growth conditions A. fumigatus (a clinical isolate) was cultured on potato dextrose agar for 4 days at 30°C. A suspension of conidia was prepared by the filtration of this culture and washed twice in 10 mM sodium phosphate buffer (NaPB; pH 7.4). In some experiments, the conidia were used as described. However, in most experiments, hyphae were studied. For this purpose, 5×104 colony-forming conidia ml−1 of yeast nitrogen base (YNB) medium supplemented with 2% glucose were cultured in flat-bottom 24-well plates for 15 h at 30°C until >95% of the conidia had germinated. Antimicrobial peptides The synthetic peptides hLF(1–11) (GRRRRSVQWCA; Mw 1,374 Da), hLF(21–31) (FQWQRNMRKVR; Mw 1,567 Da), UBI 18–35 (KVAKQEKKKKKTGRAKRR; 2,1 69 D a), U BI 29–4 1 ( T GR A KR R M Q YN R R ; 1,693 Da), and peptide 4 (part of gp120 from HIV-1; RPVVSTQLLLNGSLAEEEVV; 2,171 Da) were prepared and purified as described [7]. The peptides dhvar4 ( K R L F K K L L F S L R K Y; 1 , 8 4 1 D a ) a n d d h v a r 5 (LLLFLLKKRKKRKY; 1,848 Da) were a kind gift from Dr. W. van’t Hof (Academic Centre for Dentistry, Department of Oral Biochemistry, Free University, Amsterdam, the Netherlands). The purity of the various peptides ranged from 88–97%, as determined by reverse-phase highperformance liquid chromatography (RP-HPLC). The peptides were stored and used as previously described [7].
Eur J Clin Microbiol Infect Dis (2008) 27:1125–1129
Effects of peptides against A. fumigatus hyphae using the MTT assay To quantitate the antifungal activities of antimicrobial peptides, adherent hyphae were washed twice in NaPB before exposure for 1 h at 37°C to the various peptides. Peptide 4 was used as the negative control and amphotericin B as the positive control. Next, hyphae were washed twice and the mass of viable A. fumigatus was quantitated by a rapid colorimetric assay [12] using the tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; MTT). Hyphal damage was calculated by comparing the absorption by peptide-treated organisms with that from untreated organisms using the following equation: percentage of hyphal damage Awith peptide=amphotericin B ¼ 1 100 Awithout peptide=amphotericin B where A is the absorbance at 570 nm. For comparison of the antifungal effects of the various antimicrobial peptides against A. fumigatus, we calculated the concentration of the peptides at which 50% of the maximal antifungal activity is reached by non-linear regression with the dose–effect model according to the following equation [13]: E ¼ Emax
C EC50 þ C
where E is the observed antifungal effect at a given concentration (C) of the peptide, Emax is the maximal antifungal activity, and EC50 is the estimated concentration of the peptide at which 50% of the maximal antifungal activity is reached. Effects of peptides against A. fumigatus conidia using the AlamarBlue™ assay To compare the effects of peptides against A. fumigatus conidia, we determined their minimal inhibitory concentrations (MICs) as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) using AlamarBlue™ [14]. In short, 1 ml of a suspension of approximately 1–2×103 conidia/ml of RPMI 1640 medium with L-glutamine was pipetted together with 25 μl of AlamarBlue™ into the wells of a 24-well plate and then the various antimicrobial peptides or, as the control, no peptide were added. After incubation for 72 h at 30°C, the color change from dark blue to red was quantitated by measuring the absorbance at 600 nm. The MIC was defined as the minimal concentration of the antimicrobial peptide preventing the development of the red color.
Eur J Clin Microbiol Infect Dis (2008) 27:1125–1129
Hemolysis assay We used a hemolysis assay to establish the cytotoxicity of the different peptides. Briefly, freshly collected heparinized blood from healthy individuals was centrifuged at 100g for 15 min. The erythrocytes were washed three times with PBS, centrifuged at 1,000g for 10 min, and resuspended in PBS to a concentration of 1% (v/v). Antimicrobial peptides were serially diluted in PBS (range 1.56–600 μg/ml) and 100 μl thereof was added in triplicate to 100 μl of the erythrocyte suspension. After incubation for 1 h at 37°C, the mixtures were centrifuged at 1,000g for 5 min and 150 μl of the supernatants were transferred to a flat-bottom 96-well plate for measurement of the hemoglobin release by reading the absorbance at 450 nm. PBS and 1% Tween-20 were used to establish 0% and 100% hemolysis. The percentage of intact erythrocytes was calculated using the following formula: Apeptide APBS 1 100% ATween APBS where Apeptide is the absorbance at 450 nm in the well containing the peptide, APBS the absorbance at 450 nm in the well to which PBS was added, and ATween is the absorbance at 450 nm in the well containing Tween-20. Statistical analyses Differences between the values for cells exposed to antimicrobial peptides and those exposed to peptide 4 were analyzed using the Mann-Whitney U-test. The level of significance was set at p
